1. Field of the Invention
The present invention relates, in general, to the field of prothrombin time (PT) reagents and, in particular, to a method for manufacturing PT reagents.
2. Description of the Related Art
Blood coagulation tests may be performed for a variety of purposes, including determination of the bleeding susceptibility of patients undergoing surgery and monitoring of patients undergoing anticoagulant therapy for prevention of blood clots. A number of coagulation tests are currently in use, one of which is the “prothrombin time” (PT) test. The PT test relies upon activation of the extrinsic coagulation pathway by thromboplastin that has been added to a blood sample undergoing the PT test. Activation of the extrinsic coagulation pathway leads to the production of thrombin, a proteolytic enzyme that catalyzes the conversion of fibrinogen to fibrin, a catalysis that is essential to the clotting process.
Thromboplastin, also known as tissue factor (TF), is a membrane associated glycoprotein that forms a complex with factor VIIa. The factor VIIa/TF complex initiates the blood coagulation process. Once formed, the factor VIIa/TF complex activates a series of specific enzymes that are involved in the extrinsic and intrinsic pathways of the coagulation cascade, ultimately leading to the formation of thrombin, fibrin, platelet activation, and finally clot formation. For a related discussion, see Nemerson, Yale, Tissue Factor and Hemostasis, Blood, 71, pp.1–8 (1988).
Conventional PT tests utilize the above-described series of enzymatic events in an in vitro environment and under controlled conditions to diagnose dysfunctions or deficiencies in the blood coagulation system of patients. The time period it takes for clot formation to occur is referred to as the Prothrombin Time or PT value.
Different types of thromboplastins can either enhance or diminish the ability of a PT test to discriminate between blood samples having different prothrombin times. Thromboplastins with greater discrimination are termed “more sensitive”. The liquid phase sensitivity of a thromboplastin preparation is graded by use of the International Sensitivity Index (ISI). An ISI value can be obtained by plotting, on a logarithmic scale, the prothrombin time value obtained with a given thromboplastin lot versus the prothrombin time values obtained with a standardized reference lot of thromboplastin. The ISI value of the given thromboplastin lot is the slope of the resulting line multiplied by the ISI of the standardized reference lot of thromboplastin. More sensitive thromboplastins have lower ISI numbers around 1.0 and less sensitive thromboplastins have higher ISI numbers, typically around 2.0 to 3.0.
For use in PT testing, a highly sensitive PT reagent with an ISI of approximately 1.0 is considered most beneficial and suitable since, with an ISI of 1.0, the calculation of an International Normalized Ratio (INR) is simplified and precise. One skilled in the art will recognize that use of the INR can compensate for thromboplastin variation due to differences in sensitivity. INR is calculated using the following equation (see Hirsh et al., Oral Anticoagulants: Mechanism of Action, Clinical Effectiveness, and Optimal Therapeutic Range, Chest 2001; 119:8S–21S):INR=(patient PT/mean normal PT)ISIwhere:                patient PT=the prothrombin time of a patient's blood sample, and        mean normal PT (or MNPT)=the mean prothrombin time of blood samples from at least twenty normal (reference) sample donors.        
Conventional methods for manufacturing PT reagents generally use TF from natural or recombinant sources, natural or synthetic phospholipids, calcium and a buffer composition. U.S. Pat. No. 5,314,695, which is fully incorporated herein by reference, discloses a method for manufacturing PT reagents. The reagents include liposome compositions, in which natural or recombinant tissue factor is associated with, and inserted into, the liposomes' phospholipid bilayer. In the PT reagent manufacturing methods described therein, a determination of the quantity of TF employed in manufacturing the PT reagent is determined entirely on a mass to volume basis.
U.S. Pat. No. 5,625,036, which is fully incorporated by reference, discloses a PT reagent for use in PT testing and a method for creating lipid vesicles containing tissue factor. This PT reagent utilizes recombinant human tissue factor, phospholipids of a natural or synthetic origin, a buffer composition and calcium ions. Stabilizers and salts may also be utilized in the PT reagent.
Use of dried thromboplastin in PT test devices is described in U.S. Pat. No. 5,418,141 and U.S. patent application Ser. No. 2002/0110922, both of which are fully incorporated herein by reference. U.S. Pat. No. 5,418,141 describes the use of recombinant thromboplastins in dry reagent prothrombin time assays. The recombinant thromboplastins are employed for the purpose of improving test precision. In addition, this patent compares recombinant thromboplastin to natural thromboplastins, which, when used in dry reagent PT assays, are less sensitive and result in less precise ISI values. In U.S. patent application No. 2002/0110922, a fluidic test device is described that incorporates thromboplastin into three measurement areas, one of which is used for measuring the PT time of a blood sample. The other two measurement areas are used as control areas, thereby increasing the reliability of PT times measured using the fluidic test device. The control areas contain components in addition to thromboplastin in order to partially or completely overcome the effect of an anticoagulant present in the blood sample.
With experience, it has become apparent that conventional methods for manufacturing PT reagents do not reproducibly yield a suitably sensitive PT reagent. Still needed in the field, therefore, is a method for manufacturing a PT reagent that reproducibly yields a suitably sensitive PT reagent. In addition, the method should be simple and provide for acceptance testing of the manufactured PT reagent.